Tuning the properties of elastin mimetic hybrid copolymers via a modular polymerization method

Abstract

We have synthesized elastin mimetic hybrid polymers (EMHPs) via the step-growth polymerization of azide-functionalized poly(ethylene glycol) (PEG) and alkyne-terminated peptide (AKAAAKA)(2) (AK2) that is abundant in the cross-linking domains of the natural elastin. The modular nature of our synthesis allows facile adjustment of the peptide sequence to modulate the structural and biological properties of EMHPs. Therefore, EMHPs containing cell-binding domains (CBDs) were constructed from α,ω-azido-PEG and two types of alkyne-terminated AK2 peptides with sequences of DGRGX(AKAAAKA)(2)X (AK2-CBD1) and X(AKAAAKA)(2)XGGRGDSPG (AK2-CBD2, X = propargylglycine) via a step-growth, click coupling reaction. The resultant hybrid copolymers contain an estimated five to seven repeats of PEG and AK2 peptides. The secondary structure of EMHPs is sensitive to the specific sequence of the peptidic building blocks, with CBD-containing EMHPs exhibiting a significant enhancement in the α-helical content as compared with the peptide alone. Elastomeric hydrogels formed by covalent cross-linking of the EMHPs had a compressive modulus of 1.06 ± 0.1 MPa. Neonatal human dermal fibroblasts (NHDFs) were able to adhere to the hydrogels within 1 h and to spread and develop F-actin filaments 24 h postseeding. NHDF proliferation was only observed on hydrogels containing RGDSP domains, demonstrating the importance of integrin engagement for cell growth and the potential use of these EMHPs as tissue engineering scaffolds. These cell-instructive, hybrid polymers are promising candidates as elastomeric scaffolds for tissue engineering.

Figure 1

GPC characterization of the step growth polymerization of AK2-CBD1 (A) and EMHP-CBD1 (B) with N₃-PEG-N₃. Mobile phase: PBS, pH=7.4. Detector: photodiode array (PDA) at 214 nm. Molecular weights calculated by comparison with protein standards.

Figure 2

FTIR spectra of N₃-PEG-N₃, AK2-CBD2 peptide, and EMHP-CBD2.

Figure 3

¹H NMR spectra of AK2-CBD2 peptide (A) and EMHP-CBD2 (B) in D₂O with water suppression by presaturation.

Figure 4

Circular dichroism spectra of AK2-CDB1 peptide (A), EMHP-CBD1 (B), and AK2- CBD2 (C), and EMHP-CBD2 (D) at pH 7.4, 10, 12. All spectra were measured in PBS at 20 °C.

Figure 5

Representative loading-unloading curves for hydrated xEMHP-CBD2. Samples were allowed to recover for 1 min after each run.

Figure 6

MTT assay of N₃-PEG-N₃, AK2-CBD2 peptide, and EMHP-CBD2 multiblock at concentrations of 0.002, 0.02, 0.2, and 2 mg/mL using NHDF. An asterisk indicates statistical difference from other concentrations of the same material and a bar indicates statistical difference between two materials at the same concentration when p ≤ 0.02.

Figure 7

NHDF attachment on glass (A), xEMHP (B), and xEMHP-CBD2 (C) after 3 h in serum-free media. NHDFs were stained with CellTracker Green prior to cell seeding. Scale bar 200 μm.

Figure 8

Cell attachment (NHDFs) on glass, xEMHP, and xEMHP-CBD2 after 1h and 24h. NHDFs were stained with Draq-5 (nuclei, blue), TRITC-phalloidin (F-actin, red), and mouse anti-vinculin (primary antibody) and Alexa 488 anti-mouse (secondary antibody) (vinculin, green).

Figure 9

Proliferation of NHDFs on glass (black squares), xEMHP-CBD2 (red circles) and xEMHP free of CBDs (blue triangles) in serum-free media. Statistical analysis: an asterisk indicates statistical difference between glass and both xEMHPs, and a pound sign indicates statistical difference betwenn xEMHP-CBD2 and xEMHP at a specific time point, with p ≤ 0.01.

Scheme 1

Synthesis of EMHP-CBD1 (A) and EMHP-CBD2 (B) by step growth polymerization using azide-functionalized PEG and alkyne-terminated AK2-CBD1 (A) or AK2-CBD2 (B) peptides.